Aryl isonitriles as a new class of antimicrobial compounds

ABSTRACT

The present invention provides aryl isonitrile compounds that have antibacterial properties. More specifically, the aryl isonitrile compounds of the present invention are potent inhibitors of drug resistant strains of  Staphylococcus aureus.

BACKGROUND OF THE INVENTION

This invention is directed generally to new antimicrobial compounds andmore specifically, to aryl isonitrile compounds as a new class ofantimicrobial compounds.

Multidrug-resistant bacterial infections pose a significant globalhealth challenge afflicting more than 2 million people each year in theUnited States alone, resulting in over 23,000 fatalities. Nearly half ofthese casualties are due to infections caused by a single pathogen,methicillin-resistant Staphylococcus aureus (MRSA). Currently prevalentin the community setting, MRSA is responsible for a wide spectrum ofillnesses from superficial skin infections to invasive diseasesincluding pneumonia, osteomyelitis, and bloodstream infections. While arobust arsenal of antibiotics was once capable of treating MRSAinfections, strains of this pathogen have emerged that exhibitresistance to nearly every class of antibiotics, including agents oflast resort such as vancomycin and linezolid.

As can be seen, there is a need for need for the identification anddevelopment of novel therapeutic options capable of treating infectionsdue to MRSA.

SUMMARY OF THE INVENTION

In one aspect of the present invention there is provided an arylisonitrile compound having the general structure of compound I

wherein the compound comprises two aryl moieties linked preferably by analkyl chain, wherein n may be an integer from 1 to about 3.Alternatively, when n=0, the aryl moieties may be directly linked to oneanother. It has been shown that the critical component for antimicrobialactivity is the isonitrile substituent on one of the aryl moieties (Ar).The position of the aryl isonitrile group is not critical and may beanywhere on the aryl moiety. In another aspect of the present invention,Ar and Ar′ may be the same aryl moiety or they may be different. Ar andAr′ may be a five-membered, six-membered or seven-membered ring, or mayeven be larger. Furthermore, Ar′ may be a heteroaryl ring, comprising anoxygen or nitrogen in place of a ring carbon. Ar′ may also comprise asubstituent. The substituent may be a C1 to C3 alkyl group, a halogen,alkyloxy, trihalomethyl, or a nitro group.

These and other features, aspects and advantages of the presentinvention will become better understood with reference to the followingdrawings, description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a scheme showing the structure and synthesis of exemplarycompounds of one aspect of the present invention;

FIG. 2 is a table listing the strains of Staphylococcus aureus utilizedto test the antimicrobial properties of exemplary compounds of thepresent invention;

FIG. 3 is a table showing the minimum inhibitory concentration (MIC) ofisonitrile compounds, linezolid, and vancomycin againstmethicillin-sensitive (MSSA) and methicillin-resistant S. aureus (MRSA)strains;

FIG. 4 is a bar graph showing the cytotoxicity analysis of exemplarycompounds of the present with mammalian cells;

FIG. 5 is a table listing the calculation of physicochemical propertiesof exemplary compounds of the present invention for Lipinski's Rule of5;

FIG. 6 is a table of kinetic solubility assessment of compound 13 of thepresent invention, reserpine, tamoxifen and verapamil in PBS;

FIG. 7 is a table of permeability analysis of compound 13 of the presentinvention, ranitidine, warfarin and talinolol via the Caco-2permeability assay; and

FIG. 8 is a table of the evaluation of metabolic stability of exemplarycompound 13, verapamil and warfarin in human liver microsomes.

DETAILED DESCRIPTION OF THE INVENTION

The following detailed description is of the best currently contemplatedmodes of carrying out the invention. The description is not to be takenin a limiting sense, but is made merely for the purpose of illustratingthe general principles of the invention, since the scope of theinvention is best defined by the appended claims.

Broadly, the present invention provides novel aryl isonitrile compounds,wherein the compounds comprise two aryl moieties linked by an alkylchain and wherein one of the aryl groups comprises an isonitrilesubstituent. Methods of use are also provided for the treatment of drugresistant bacteria, in particular, drug-sensitive and drug-resistantStaphylococcus aureus, commonly referred to as MRSA or MSSA. For theease of description, MRSA will be used to refer to all strains ofdrug-sensitive and drug resistant S. aureus.

The compounds of the present invention are able to inhibit bacterialgrowth at micromolar concentrations while showing no toxicity tomammalian cells. This is in contrast to the state of the art, whereinthere is a lack of new antibiotic compounds to treat MRSA infections.

In one aspect of the present invention there is provided an arylisonitrile compound having the general structure of compound I

wherein the compound comprises two aryl moieties linked preferably by analkyl chain, wherein n may be an integer from 1 to about 3.Alternatively, when n=0, the aryl moieties may be directly linked to oneanother. It has been shown that the critical component for antimicrobialactivity is the isonitrile substituent on one of the aryl moieties (Ar).The position of the aryl isonitrile group is not critical and may beanywhere on the aryl moiety. In another aspect of the present invention,Ar and Ar′ may be the same aryl moiety or they may be different. Ar andAr′ may be a five-membered, six-membered or seven-membered ring, or mayeven be larger. Furthermore, Ar′ may be a heteroaryl ring, comprising anoxygen or nitrogen in place of a ring carbon. Ar′ may also comprise asubstituent. The substituent may be a C1 to C3 alkyl group, a halogen,alkyloxy, trihalomethyl, or a nitro group.

In another aspect of the present invention, the alkyl chain may be analkane or an alkene. Preferably the alkyl chain, or bridge, between thetwo aryl moieties is an alkene. It has been shown (see Example) thatcompounds comprising an alkane bridge have a significantly lowerantibiotic activity against MRSA than compound with an alkene bridgebetween the two aryl moieties, Ar and Ar′. However, the compounds withan alkane bridge did have antibiotic activity against several MRSAstrains.

In a further aspect of the present invention, the alkyl bridge maycomprise a substituent. The substituent may be hydrogen, a C1 to C3alkyl group, a halogen, alkyloxy, trihalomethyl, or a nitro group.

In another aspect of the present invention, there is provided an arylisonitrile compound having the structure of compound II or III:

wherein R is an alkyl, a cycloalkyl, a heteroalkyl, or an aryl group;n is an integer from 0 to 4;X is hydrogen, a C1 to C3 alkyl group, a halogen, alkyloxy,trihalomethyl, or a nitro group;W, Y and Z are each independently —CH—, —N— or —O—; andpharmaceutically acceptable salts thereof.

Exemplary compounds of the present invention having structures II or IIIare given in FIG. 1. In an exemplary embodiment, R is a methyl group andn is an integer from 0 to 4. Alternatively, compounds II and III maylack substituent R on the alkyl bridge. In another aspect of the presentinvention, X may be a hydrogen, a C1-C3 alkyl group (i.e. methyl, ethyl,n-propyl or n-butyl), a halogen, such as, but not limited to fluorine(F), an alkoxy such as, but not limited to methoxy, a trihalomethyl suchas but not limited to trifluoromethyl or a nitro group.

In another aspect of the present invention, methods are providing forinhibiting MRSA by treating the MRSA with the compounds of the presentinvention. Exemplary strains of MRSA that may be inhibited by thecompounds of the present invention are provided in FIGS. 2 and 3. Themethods may comprise the steps of contacting the MRSA with an inhibitoryamount of the compounds of the present invention. In one aspect of thepresent invention, the compounds may be from about 50 μM to about 100μM.

Alternatively, methods are provided for treating a patient with MRSAcomprising administering a therapeutic amount of the compounds of thepresent invention to a patient having MRSA. It will be appreciated thatthe amount of compound to be administered with depend on the strain ofMRSA and the severity of the infection. The amount may be determined bythe skilled practitioner without undue experimentation. The compoundsmay be administered in a manner consistent with treatment of a MRSAinfection. In one exemplary aspect, the compounds are administeredsystemically in a pharmaceutically acceptable carrier. In anotherexemplary aspect of the present invention, the compounds may beadministered topically. This would be desirable if the MRSA infection isa skin infection. Moreover, the compounds of the present invention maybe administered along with other compounds to treat the MRSA infection.

Example

A whole-cell screening of a small number of in house generated smallmolecules with diverse structural skeletons (about 250 molecules) hasbeen conducted against MRSA USA300 with the aim to identify compoundswith novel skeletons to target antibiotic drug resistance. Among severalhit molecules revealed by this mini-screening effort, compound 1 with anisonitrile group attached to a stilbene system was shown to be capableof inhibiting bacterial growth at a concentration of 32 μM (FIG. 1). Thepresence of an isonitrile moiety in this compound is quite unique giventhat few antimicrobial compounds possessing the isonitrile moiety intheir core structure have been described in literature and those thathave been are natural products. Marconi, G. G. et al., J. Antibiot.1978, 31, 27-32; Mo, S. et al., J. Nat. Prod. 2009, 72, 894-899; Raveh,A. et al., J. Nat. Prod. 2007, 70, 196-201; Sugawara, T. et al., J.Antibiot. 1997, 50, 944-948; Fujiwara, A. et al., Agric. Biol. Chem.1982, 46, 1803-1809. Marquez, J. A. et al., J. Antibiot. 1983, 36,1101-1108. The novel structural skeleton of compound 1 as anantibacterial compound against drug resistant strains prompted furtherstudy of these types of isonitrile compounds. Herein is reported thechemical synthesis, structure-activity relationship study, andevaluation of the antibacterial performance of compound 1 and closelyrelated analogs against several clinically-relevant MRSA and VRSAstrains. These efforts have led to the identification of more potentcompounds with MIC as low as 2 μM but do not show any cytotoxicityagainst mammalian cells up to a concentration of 64 μM. Physiochemicalanalysis of several potent lead compounds has been described to guidethe next stage of developing these promising compounds into theantibiotic drug pipeline.

Chemistry:

In general, the stilbene isonitrile analogs were prepared from benzylicbromide 2 (FIG. 1) which was converted to phosphonate 3 byMichaelis-Arbuzov reaction. (Abruzov, B. A. et al., Pure. Appl. Chem. 9(1964) 307-353. The nitro group of 3 was then converted to an isonitrilegroup upon a sequence of hydrogenation and Hofmann isonitrile synthesisusing dichlorocarbene. Weber W. P. & Gokel G. W., Tetrahedron Lett.(1972) 1637-1640. Compound 4 then served as a divergent point tosynthesize a collection of analogs with a Horner-Wadsworth-Emmonsreaction. Zhang, B. & Studer, A., Org. Lett. 2014, 16, 1216-1219. Bytreating various ketones and aldehydes with stabilized phosphonatecarbanions derived from phosphonates 4, thirty-three stilbene isonitrileanalogs (1, 5-25 and 27-37) and one styrene isonitrile analog (26) wereobtained. This collection also included compounds with the isonitrilegroup at different positions on the aromatic ring as well as pyridinecontaining analogs. In order to investigate the importance of theisonitrile group for the observed biological activity, compoundscontaining a hydrogen atom (42) or a nitrile group (43) at theisonitrile-substitution position were prepared as well using theHorner-Wadsworth-Emmons reaction. Additionally, four biaryl isonitrileanalogs (46 and 49-51) were prepared. Zhang, B. et al., Angew. Chem.Int. Ed. 2013, 52, 10792-10795. Compound 46 was prepared fromcommercially available amine 44 via form-amide formation followed bydehydration. Compounds 49-51 were synthesized from 2-bromoanilinederivatives (47) and aryl-boronic acids. Suzuki cross-coupling converted47 to biaryl amines 48 smoothly. The latter was then converted to 49-51via the aforementioned formamide formation and dehydration sequence.Lastly, compound 53 was prepared with a saturated two-carbon chain toinvestigate the importance of the double bond linker between the twoaromatic moieties. All the newly synthesized compounds were purifiedusing flash chromatography before entering biological evaluations.

Biological Results and Discussion:

Antimicrobial Susceptibility Analysis of the Isonitrile CompoundsAgainst Clinically Relevant Isolates of MRSA.

The bacterial growth inhibiting activity of these synthetic analogs ofhit compound 1 were subsequently evaluated (FIG. 3). When thesederivatives were screened against MRSA via the broth microdilutionassay, the results revealed several interesting structural elements thatappeared to play an important role in the antimicrobial activity ofthese compounds. Initial inspection of the structural moieties of 1revealed that the presence of an isonitrile group was essential for itsantimicrobial activity. When the isonitrile group of 1 (MIC against MRSAranging from 8-64 μM) was removed (as in compound 42), a complete lossin the anti-MRSA activity of 42 was observed (MIC>128 μM). A similarpattern was observed when reviewing the MIC results for compounds 13 and43. Compound 13, one of the most potent derivatives constructed (withMIC values against MRSA as low as 2 μM), contains the isonitrile group;when the isonitrile group of 13 is replaced with an isosteric nitrilegroup (resulting in compound 43), complete loss of antimicrobialactivity was observed. Similarly, compound 53 with an isonitrile groupis active against several strains evaluated particularly MRSA USA100,MRSA USA300, MRSA NRS119, and VISA NRS1, while compound 52 without theisonitrile group lacks antimicrobial activity. These results confirmthat the isonitrile group appears necessary for these compounds topossess activity against MRSA and may play an important role in bindingto the compound's molecular target.

The presence of a second aromatic substituent (connected to theisonitrile-phenyl group) also appeared critical to the biologicalactivity observed; replacement of this moiety in 1 with a diethylphosphonate (as in analogue 4a with an ortho-isonitrile group) resultedin complete loss of activity against MRSA (MIC>128 μM). Likewise,substitution of this second aromatic substituent with a cycloalkane (cf.26) rendered this compound inactive against several MRSA isolates(including MRSA USA300, MRSA USA500, and MRSA NRS119). The presence ofan alkene bridge between the two aromatic substituents in 1 alsoappeared to be important. When the alkene bridge between the twoaromatic substituents was removed, as in compound 46, the compoundlacked activity against three strains of MRSA (USA300, USA500, andNRS119). A similar loss in antimicrobial activity was observed withcompounds 49 and 50 indicating that the stilbene isonitrile core of 1plays an important role in its antimicrobial activity. This notion wasfurther supported by a direct comparison of compounds 13 and 53.Compound 53 is a saturated analog of compound 13 and contains a flexibletwo-carbon linker between the two aromatic moieties. In general,compound 53 is less potent than compound 13 against all the strainstexted except for VISA NRS1.

It was then evaluated how substituents on the double bond would affectthe antimicrobial activity. Interestingly, removal of the ethyl group of1 (cf. 13) resulted in a dramatic improvement in antimicrobial activity(a two-to-eight fold reduction in the MIC against MRSA was observed).When the ethyl group was replaced by methyl (5), n-propyl (6), n-butyl(7) and phenyl (8) groups, a noticeable change in the MIC value forthese compounds was observed.

It was next assessed how substituents on the non-isonitrile-containingaromatic ring would affect the potency against MRSA. Analoguesconstructed included substitution of methoxy group (14-16), fluoride(17-19), trifluoromethyl group (20-22), methyl group (23), n-butyl group(24), and nitro group (25). Interestingly most of these modificationsdid not produce a major improvement in the MIC observed against MRSA,when compared to the activity of 13. Additionally the positioning ofthese groups around the benzene ring did not appear to have an impact onthe antimicrobial activity of the compound. While most of thesemodifications had little effect on improving the antimicrobial activityof these compounds, one substitution had an observed deleterious effect.Compound 24, containing a n-butyl group, lacked activity against mostMRSA strains tested (MIC>128 μM); interestingly, 23, with a methyl groupwas active against all MRSA strains tested albeit at a higherconcentration than 13 (MIC of 23 ranges from 4 to 64 μM against MRSA).This would appear to indicate that the presence of an alkyl group (inparticular one of increased length) is undesirable and can have anegative effect on the activity of these compounds against MRSA. Analogscontaining a pyridine ring were synthesized and tested as well (27, 30,and 33) and reduced antimicrobial activities were observed.

All the analogs discussed above contain an ortho-substituted isonitrilegroup. It was wondered how the relative position of the isonitrile groupwould affect the antimicrobial activity and prepared eight analogs withthe isonitrile group in para- and meta-relationship to the double bond(cf. 28, 29, 31, 32, 34-37). Different antimicrobial activity patternsare observed. For the group of 13, 36, and 37, the ortho-substitutedcompound 13 is still the most potent one against most of the strainstested and slight improvement was observed for the para-substitutedcompound 37 against MSSA (NRS72) and MRSA USA500. Interestingly, for thegroup of 33, 34, and 35, the para-substituted compound 35 is much moreactive against all the strains tested than the ortho- andmeta-substituted ones. The groups of 27-29 and 30-32 are less potentthan the aforementioned two groups, which indicate that the position ofthe nitrogen atom in the pyridine ring is important for the observedantimicrobial activity as well.

After completing a preliminary examination of the structure-activityrelationship of these compounds, it was assessed whether these compoundswould retain their activity against several of the most challengingstrains of MRSA (FIGS. 2 and 3). When tested against an array ofclinically relevant MRSA isolates, the most potent compounds (6, 8-18,20-21, 25 and 37) did retain their antimicrobial activity. Indeed thesecompounds possessed potent activity against MRSA isolates prevalent inthe healthcare setting such as MRSA USA100 (responsible for invasivediseases in infected hospitalized patients), and MRSA USA200 (associatedwith more severe morbidity in affected patients due to the production oftoxins that can lead to toxic shock syndrome). In addition to this,these compounds exhibited potent activity against MRSA USA300, a strainthat has been linked to the majority of MRSA skin and soft tissueinfections present in the community setting. Furthermore, thesecompounds demonstrated strong antimicrobial activity against MRSAstrains exhibiting resistance to numerous antibiotic classes includingpenicillins, aminoglycosides (NRS1, USA200, and USA500), macrolides(USA100, USA200, USA300, USA500, and USA700), lincosamides (USA100,USA200, USA500), tetracyclines (NRS1, USA300, and USA500), andfluoroquinolones (USA100 and USA500). Additionally, compounds 10, 11,12, 21, 25, 32 and 35 exhibited potent antimicrobial activity (MICbetween 2 and 16 μM) against clinical isolates of S. aureus exhibitingresistance to antibiotics deemed agents of last resort, namelyvancomycin (VRS2). These results indicated cross-resistance betweenthese antibiotics and the aryl isonitrile compounds is unlikely; thislends further credence to the notion that the aryl isonitrile compoundsmay have potential to be developed as future alternatives to theseantibiotics.

Toxicity Analysis of Most Potent Aryl Isonitrile Compounds AgainstMammalian Cells.

Identification of compounds exhibiting potent antimicrobial activity isthe first step in a lengthy process for drug development. However, manycompounds with promising antimicrobial activity fail to advance furtherin this process due to concerns about toxicity to mammalian tissues.Selective toxicity is a critical feature novel antimicrobial compoundsmust possess. The ability for antimicrobial agents to exhibit theiractivity on the target microorganism while not causing harm to host(mammalian) tissues is important to ascertain early in the drugdiscovery process. To determine if compound 1 and fifteen of its mostpotent derivatives against MRSA exhibited toxicity to mammalian tissues,these compounds were screened against a murine macrophage (J774) cellline utilizing the MTS assay (FIG. 4). Initial inspection of thestructure-activity relationship revealed that the isonitrile moietyappeared to be a vital component in the antimicrobial activity of thesecompounds. This was a point of concern given the isonitrile group hasbeen associated with a high degree of toxicity in certain compoundspresent in nature. Goda, M. et al., J. Biol. Chem. 2001, 276,23480-23485. However, when the most potent compound, 13 (containing theisonitrile moiety), and its analogue 42 (lacking the isonitrile moiety)were tested against J774 cells, they produced identical results (neithercompound was toxic up to a concentration of 64 μM). This would indicatethat the isonitrile group in these compounds does not contribute toundesirable toxicity to mammalian cells. This result is similar to astudy conducted at Bayer AG that found compounds, in their discoverypipeline, containing the isonitrile moiety were not toxic to mice whenadministered orally or subcutaneously (even at concentrations in excessof 500 mg/kg). Ugi, I. et al., Angew. Chem. Int. Ed. 1965, 4, 472-484.In addition to this, at a concentration of 32 μM, all of the compoundstested, with the exception of 25 with a nitro group, were not toxic.When the compounds were tested at a concentration of 64 μM, thirteen outof seventeen compounds were found to not be toxic to J774 cells.Compounds 11, 12, 19, and 25 were found to be toxic at 64 μM. When thecompounds were tested at 128 μM, all compounds were found to be toxicwith the exception of compounds 15, 30 and 37. For the most activecompounds (such as 13), a 16-to-32-fold difference exists between theconcentrations at which the compounds exhibit anti-MRSA activity (MIC)compared to the concentration where toxicity is observed.

Preliminary Study of Physicochemical Properties of the IsonitrileCompounds Using Lipinski's Rule of 5, Kinetic Solubility Analysis, andCaco-2 Permeability Assay.

After confirming twelve isonitrile compounds exhibited strongantimicrobial activity against MRSA and were not toxic to mammaliancells up to a concentration of 64 μM, the physicochemical properties ofnine representative compounds was analyzed. These properties play animportant role in determining the appropriate route of administration(i.e. systemic vs. local) by which compounds with biological activitycan be delivered to the host. Kerns, E. H. & Di, L. Drug-likeproperties: concepts, structure design and methods—from ADME to toxicityoptimization. Amsterdam; Boston: Academic Press; 2008. Additionally, thephysicochemical properties of a compound will have a direct impact onits pharmacokinetic profile (in particular absorption and metabolism),providing important information on the likelihood of attaining successin translating a promising compound into a viable drug candidate.Compounds possessing a limited physicochemical profile can have issuespertaining to solubility (limiting the ability of a drug to be absorbedfrom the intestinal tract) and permeability which can hinder acompound's ability to cross biological membranes, reach the bloodstream,and arrive at the target site of an infection (thus limiting their usesystemically). Analysis of the hydrogen bonding potential andlipophilicity of promising compounds can lend valuable insight intoprobable issues pertaining to solubility and permeability. UtilizingLipinski's Rule of 5 (Lipinski, C. A. et al., Adv. Drug Deliv. Rev.2001, 46, 3-26) and topological polar surface area (tPSA) as guidelines,it was predicted whether the most promising isonitrile compoundsdisclosed herein would have suitable physicochemical properties to allowthem to be used in systemic applications. Compounds violating more thanone principle in the Rule of 5 would be expected to have issuespertaining to solubility and permeability.

As depicted in the table of FIG. 5, seven out of nine compounds analyzed(including the most promising compound 13) possessed calculated log P (clog P) and tPSA values that would indicate that these compounds shouldnot experience issues pertaining to both solubility and permeability.Two derivatives (8 and 12) possessed a c log P value above five, whichis a violation to the Rule of 5. This particular violation would suggestdifficulty in the ability of these compounds to passively cross abiological membrane. No significant difference is observed in the numberof hydrogen bond donor (zero) and acceptor (1-2) groups present in thestructure of the compounds analyzed, which would indicate that theywould be expected to share a similar solubility profile. As compound 13was the most promising compound identified thus far (with MIC values aslow as 2 μM against MRSA, exhibiting no toxicity to mammalian cells upto a concentration of 64 μM, and possessing zero violations to the Ruleof 5), this analogue was selected for further analysis.

To confirm if the prediction of 13 having a good physicochemical profilewas accurate, a kinetic solubility screen (using phosphate-bufferedsaline) and Caco-2 permeability analysis was performed with thiscompound. The solubility screen determined the highest concentration 13and three control drugs were capable of being fully dissolved in anaqueous solvent (PBS). As presented in the table of FIG. 6, thisexperiment revealed that compound 13 possessed partial aqueoussolubility (soluble up to 15.6 μM), identical to the control drugsreserpine and tamoxifen.

The Caco-2 permeability assay revealed that compound 13 was not able topermeate across the Caco-2 bilayer. As presented in the table in FIG. 7,this compound was unable to cross from the apical (A) to basolateral (B)surface of the membrane (apparent permeability, Papp=0.0 cm/sec). Asimilar pattern was observed in the basolateral to apical direction withPapp=0.0 cm/sec (indicating this compound is unlikely a substrate for anefflux transporter, like talinolol, which would be one plausibleexplanation for the inability of this compound to traverse themembrane). This is in stark contrast to the control drug warfarin, whichis able to effectively permeate across the membrane from the basolateralto apical surface (Papp=27.0×10-6 cm/sec). This result is a bitsurprising given the size, structure, and calculated partitioncoefficient (c log P=4.107) for 13. Thus, in addition to possessing onlypartial aqueous solubility, 13 also possesses a poor permeabilityprofile, indicating that, in its present state, this compound would notbe suitable for use systemically.

The result from the Caco-2 permeability analysis is in agreement withthe overall result obtained from the kinetic solubility screenindicating that 13, though a promising antimicrobial candidate, needs toundergo further structural modifications to enhance its physicochemicalprofile in order for it to be used systemically. In addition tomodifying the structure of this compound, formulation technology can beutilized to overcome this compound's current limitations. Thistechnology has been used to improve the drug-like properties ofpromising compounds with similar kinetic profiles to 13 in order topropel these compounds into further stages of drug development. By usinga spray drying dispersion technique (Kwong, A. D. et al., Nat.Biotechnol. 2011, 29, 993-1003), the antisolvent crystallization method(Lonare, A. A. & Patel, S. R., Int. J. Chem. Eng. Appl. 2013, 4,337-341), or combining the active compound with an excipient to createan amorphous solid dispersion (Van den Mooter, G., Drug Discov. Today.Technol. 2012, 9, e71-e174), the aqueous solubility, permeability andbioavailability profile of this compound may be significantly improved.Identifying that 13 has a problematic physicochemical profile early inthe drug discovery process will permit medicinal chemists andformulation scientists to invest time and effort to enhancing both thephysiochemical and pharmacokinetic profiles of this promising newantimicrobial compound.

Metabolic Stability Analysis of 13 Via Microsomal Stability Analysis.

In addition to studying the solubility and permeability profile ofcompound 13, the stability of this compound to metabolic processespresent in the liver was investigated using human liver microsomes (FIG.8). Drugs administered systemically often are subject to variousmetabolic processes that can convert the active compound to inactivemetabolites. Pharmaceutical compounds that are slow to be metabolizedhave multiple advantages including an improved pharmacokinetic profile,reduced frequency of doses that need to be given to patients (leading tobetter patient compliance), while also ensuring the active drugcirculates within the patient's system to assist with treating andclearing an infection. As the liver is the primary organ for metabolismof drugs administered systemically in the body, incubating compoundswith liver microsomes can shed valuable insight into the stability ofthese compounds to metabolic processes.

When 13 was incubated with human liver microsomes, it was found to berapidly metabolized (only 24% of the parent compound remained after onehour) similar to the highly metabolized control drug, verapamil (13%remained after one hour incubation with liver microsomes) (FIG. 11).While verapamil appeared to be metabolized via a NADPH-mediated process(as 94% of the drug remained after one hour when the co-factor NADPH wasremoved from the reaction mixture), 13 does not appear to mimic thisresult as only 51% of the parent compound remained after one hour whenNADPH was not present. This would appear to suggest that 13 ismetabolized by more than one enzyme system/reaction (one dependent onthe co-factor NADPH (most likely the cytochrome P450 system), and oneindependent of NADPH). The metabolic stability analysis performed lendsfurther credence to the argument that in their present state, 13, wouldnot be suitable for use in systemic applications to treat MRSAinfections.

Methods: Synthetic Procedures and Spectra Data

General Methods.

Reactions were performed using standard syringe techniques under argonunless stated otherwise. Starting materials and reagents were used asreceived from suppliers (Aldrich, Alfa Aeser, Acros). Anhydrous THF wasdistilled over sodium benzophenone under argon. Acetonitrile (CH3CN),dichloromethane (CH2Cl2), methanol (MeOH), and toluene were purified bypassing the previously degassed solvents through activated aluminacolumns. Flash chromatography was performed using silica gel (230-400mesh). Thin layer chromatography (TLC) was performed using glass-backedsilica plates (Silicycle). NMR spectra were recorded on a BrukerARX-300, Bruker ARX-400 spectrometer, DRX-500 or AV-500 spectrometer atroom temperature. Chemical shifts (in ppm) are given in reference to thesolvent signal [1H NMR: CDCl3 (7.26); 13C NMR: CDCl3 (77.2).]. 1H NMRdata are reported as follows: chemical shifts (δ ppm), multiplicity(s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet, br=broad),coupling constant (Hz), and integration. 13C NMR data are reported interms of chemical shift and multiplicity. IR data were recorded on aThermo Nicolet Nexus 470 FTIR.

A Representative Procedure for the Synthesis of Aryl Isonitriles:(E)-1-isocyano-2-(2-phenylbut-1-en-1-yl)benzene (1)

To a stirred solution of diisopropyl amine (52 mg, 0.52 mmol) in THF(1.3 ml) was added a solution of n-BuLi (2.5 M in hexane, 0.174 ml, 0.43mmol) dropwise at −78° C. After stirring for 5 min, a solution ofdiethyl (2-isocyanobenzyl)phosphonate 4 (100 mg, 0.395 mmol) in THF (1ml) was added dropwise at −78° C. The resulting solution was stirred foran additional 30 min and a solution of propiophenone (48 mg, 0.36 mmol)in THF (1 ml) was added dropwise. The reaction was stirred for anadditional 30 min at −78° C. then warmed to room temperature and stirredfor 1 h. A saturated aqueous ammonium chloride solution (4 ml) and Et2O(4 ml) were added. The aqueous layer was extracted with Et2O (3×5 ml)and the combined organic layers were washed with brine (10 ml), driedover anhydrous sodium sulfate and concentrated under reduced pressure.The resulting residue was purified by flash chromatography(CH2Cl2/hexane=1/4) to yield(E)-1-isocyano-2-(2-phenylbut-1-en-1-yl)benzene 1 (50 mg, 60% yield.)

¹H NMR (300 MHz, CDCl3) δ 7.35-7.17 (m, 4H), 7.20-6.99 (m, 3H), 6.96(td, J=7.7, 1.4 Hz, 1H), 6.78 (dd, J=7.9, 1.5 Hz, 1H), 6.63 (s, 1H),2.62 (qd, J=7.4, 1.5 Hz, 2H), 1.14 (t, J=7.4 Hz, 3H); ¹³C NMR (100 MHz,CDCl3) δ 166.1, 149.1, 140.3, 134.7, 130.2, 128.3 (3C), 128.2, 127.2,126.7, 126.5, 119.7, 33.0, 12.8.

Spectra Data of New Aryl Isonitriles:(E)-1-isocyano-2-(2-phenylhex-1-en-1-yl)benzene (7)

¹H NMR (500 MHz, CDCl3) δ 7.66-7.19 (m, 4H), 7.19-7.04 (m, 3H), 6.95(td, J=7.7, 1.3 Hz, 1H), 6.75 (d, J=10 Hz, 1H), 6.60 (s, 1H), 2.59 (t,J=7.5 Hz, 2H), 1.32-1.56 (m, 4H), 0.91 (t, J=7 Hz, 3H); ¹³C NMR (125MHz, CDCl3) δ 165.9, 147.8, 140.3, 134.9, 130.3, 128.5 (2C), 128.4,128.3, 127.3, 126.8, 126.6, 120.8, 39.8, 30.0, 22.2, 13.9; IR (neat):2956, 2925, 2854, 2117, 1478, 1448 cm⁻¹; MS (ESI): m/z=284.14 calc. forC19H19N[M+Na]+, found 284.24.

(2-(2-isocyanophenyl)ethene-1,1-diyl)dibenzene (8)

¹H NMR (500 MHz, CDCl3) δ 7.38-7.28 (m, 8H), 7.16-7.11 (m, 3H), 7.10 (s,1H), 7.00 (dt, J=8.2, 1.1 Hz, 1H), 6.85 (d, J=8.0 Hz, 1H); ¹³C NMR (125MHz, CDCl3) δ 166.5, 146.7, 142.4, 139.5, 134.6, 130.4, 130.2, 128.5,128.4, 128.3 (3C), 128.1, 127.9, 127.3, 126.9, 122.0; IR (neat) 3059,3023, 2923, 2853, 2116, 1491, 1473, 1443, 1280, 1114, 1027, 942, 885cm⁻¹; MS (ESI): m/z=304.11 calc. for C21H15N[M+Na]+, found 304.24.

(E)-1-(2-(4-fluorophenyl)but-1-en-1-yl)-2-isocyanobenzene (9)

¹H NMR (500 MHz, CDCl3) δ 7.30 (dd, J=7.9, 1.3 Hz, 1H), 7.10 (t, J=7.5Hz, 1H), 7.07-7.04 (m, 2H), 7.00 (t, J=7.8 Hz, 1H), 6.94 (m, 2H), 6.76(d, J=8.0 Hz, 1H), 6.59 (s, 1H), 2.58 (qd, J=7.4, 1.4 Hz, 2H), 1.11 (t,J=7.4 Hz, 3H); ¹³C NMR (125 MHz, CDCl3) δ 166.0 162.0 (d, J=244 Hz),148.0, 136.1 (d, J=4 Hz), 134.6, 130.3, 130.1 (d, J=8 Hz), 128.4, 126.9,126.6, 125.6, 120.2, 115.4 (d, J=21 Hz), 32.9, 12.8; IR (neat) 2967,2930, 2117, 1602, 1507, 1222, 1178, 879, 836 cm⁻¹; MS (ESI): m/z=274.10calc. for C17H14FN[M+Na]+, found 274.16.

(E)-1-isocyano-2-(2-(4-(trifluoromethyl)phenyl)but-1-en-1-yl)benzene(10)

¹H NMR (500 MHz, CDCl3) δ 7.51 (d, J=8.0 Hz, 2H), 7.31 (dd, J=8.0, 1.3Hz, 1H), 7.22 (d, J=8.0 Hz, 2H), 7.12 (td, J=7.7, 1.4 Hz, 1H), 7.01 (td,J=7.7, 1.3 Hz, 1H), 6.73 (d, J=8.1 Hz, 1H), 6.67 (s, 1H), 2.61 (qd,J=7.4, 1.5 Hz, 3H), 1.12 (t, J=7.4 Hz, 3H); ¹³C NMR (125 MHz, CDCl3) δ166.1, 147.6, 144.1, 134.1, 130.1, 128.7 (2C), 128.5, 127.2, 126.6,126.1 (q, J=269 Hz), 125.30, 125.27, 121.1, 32.6, 12.7; IR (neat) 2968,2931, 2118, 1322, 1164, 1109, 1066, 881, 843 cm⁻¹; MS (ESI): m/z=324.10calc. for C18H14F3N[M+Na]+, found 324.08.

(E)-1-isocyano-2-(2-(3-(trifluoromethyl)phenyl)but-1-en-1-yl)benzene(12)

¹H NMR (400 MHz, CDCl3) δ 7.49 (dd, J=8.3, 1.1 Hz, 1H), 7.38-7.26 (m,4H), 7.12 (td, J=7.7, 1.4 Hz, 1H), 7.00 (td, J=7.7, 1.4 Hz, 1H),6.67-6.72 (m, 2H), 2.63 (qd, J=7.4, 1.5 Hz, 2H), 1.14 (t, J=7.4 Hz, 3H);¹³C NMR (125 MHz, CDCl3) δ 166.6, 147.7, 141.3, 134.4, 132.2, 131.1,130.9, 130.4, 129.1, 128.7, 127.5, 126.9, 126.2 (q, J=267 Hz), 125.4,124.3, 121.4, 32.6, 12.8; IR (neat) 2969, 2118, 1324, 1202, 1163, 1072,903, 828 cm⁻¹; MS (ESI): m/z=324.10 calc. for C18H14F3N[M+Na]+, found324.08.

(E)-1-isocyano-2-styrylbenzene (13)

¹H NMR (400 MHz, CDCl3) δ 7.75 (dd, J=8.4, 1.3 Hz, 1H), 7.64-7.55 (m,2H), 7.49-7.13 (m, 8H); ¹³C NMR (100 MHz, CDCl3) δ 167.0, 136.4, 133.7,132.7, 129.4, 128.8, 128.6, 128.0, 127.3, 127.0, 125.4, 125.0, 122.2; IR(neat) 3044, 3022, 2119, 1632, 1598, 1480, 1446, 1288, 1263, 1221, 1198,1092, 959, 875 cm⁻¹; MS (ESI): m/z=228.08 calc. for C15H11N[M+Na]+,found 228.00.

(E)-1-isocyano-2-(4-methoxystyryl)benzene (14)

¹H NMR (500 MHz, CDCl3) δ 7.71 (dd, J=7.5, 1.2 Hz, 1H), 7.56-7.50 (m,2H), 7.41-7.36 (m, 2H), 7.27-7.11 (m, 3H), 6.96-6.89 (m, 2H), 3.85 (s,3H); ¹³C NMR (125 MHz, CDCl3) δ 166.7, 160.0, 134.1, 132.2, 129.4,129.2, 128.4, 127.5, 127.3, 127.0, 125.2, 112.0, 114.3, 55.4; IR (neat)2924, 2843, 2122, 1633, 1604, 1511, 1481, 1270, 1253, 1172, 1091, 961,868 cm⁻¹; MS (ESI): m/z=258.09 calc. for C16H13NO[M+Na]+, found 258.10.

(E)-1-isocyano-2-(3-methoxystyryl)benzene (15)

¹H NMR (500 MHz, CDCl3) δ 7.73 (d, J=7.9 Hz, 1H), 7.42-7.36 (m, 3H),7.32-7.26 (m, 2H), 7.19 (s, 1H), 7.16 (d, J=7.8 Hz, 1H), 7.10 (m, 1H),6.88 (dd, J=8.2, 2.4 Hz, 1H), 3.86 (s 3H); ¹³C NMR (125 MHz, CDCl3) δ167.0, 159.9, 137.8, 133.6, 132.6, 129.8, 129.4, 128.0, 127.3, 125.5,124.9, 122.5, 119.6, 114.2, 112.2, 55.3; IR (neat) 3072, 3034, 2119,1607, 1581, 1489, 1482, 1448, 1286, 1239, 1157, 1135, 1093, 941, 873cm⁻¹; MS (ESI): m/z=258.09 calc. for C16H13NO[M+Na]+, found 258.00.

(E)-1-isocyano-2-(2-methoxystyryl)benzene (16)

¹H NMR (500 MHz, CDCl3) δ 7.80 (d, J=8.0 Hz, 1H); 7.66 (d, J=7.6 Hz,1H), 7.57 (d, J=16.4 Hz, 1H), 7.46 (d, J=16.5 Hz, 1H), 7.40-7.37 (m,2H), 7.31 (t, J=8.2 Hz, 1H), 7.24 (d, J=8.0 Hz, 1H), 7.00 (t, J=7.6 Hz,1H), 6.93 (d, J=8.2 Hz, 1H), 3.91 (s, 3H); ¹³C NMR (125 MHz, CDCl3) δ166.6, 157.2, 134.3, 129.6, 129.2, 127.6, 127.5, 127.1 (2C), 125.4 (2C),124.8, 122.5, 120.8, 110.9, 55.4; IR (neat) 3033, 2958, 2936, 2834,2119, 1489, 1463, 1334, 1242, 1191, 1107, 1091, 1031, 992, 965, 879cm⁻¹; MS (ESI): m/z=258.09 calc. for C16H13NO[M+Na]+, found 258.10.

(E)-1-(4-fluorostyryl)-2-isocyanobenzene (17)

¹H NMR (500 MHz, Chloroform-d) δ 7.72 (dd, J=8.4, 1.3 Hz, 1H), 7.58-7.52(m, 2H), 7.40-7.37 (m, 2H), 7.33 (d, J=16.3 Hz, 1H), 7.28 (td, J=7.6,1.1 Hz, 1H), 7.16 (d, J=16.3 Hz, 1H), 7.10-7.06 (m, 2H); ¹³C NMR (125MHz, CDCl3) δ 167.0, 162.9 (d, J=287 Hz), 133.5, 132.6 (d, J=3 Hz),131.4, 129.4, 128.61, 128.55, 128.0, 127.3, 125.3, 121.9, 115.8 (d, J=22Hz); IR (neat) 3045, 2926, 2122, 1567, 1508, 1479, 1265, 1232, 1177,1159, 960, 933, 817 cm⁻¹; MS (ESI): m/z=224.09 calc. for C15H10FN[M+H],found 224.08.

(E)-1-fluoro-2-(2-isocyanostyryl)benzene (18)

¹H NMR (500 MHz, CDCl3) δ 7.77 (dd, J=8.0, 1.3 Hz, 1H), 7.70 (td, J=7.7,1.7 Hz, 1H), 7.48 (d, J=16.5 Hz, 1H), 7.42-7.37 (m, 3H), 7.30-7.27 (m,2H), 7.18 (td, J=7.6, 1.2 Hz, 1H), 7.10 (m, 1H); ¹³C NMR (125 MHz,CDCl3) δ 167.2, 160.5 (d, J=249 Hz), 133.6, 129.9 (d, J=8 Hz), 129.4,128.3, 127.2, 127.17 (d, J=3 Hz), 125.5, 124.7 (d, J=3 Hz), 125.0,124.4, 124.3, 124.1 (d, J=3 Hz), 115.8 (d, J=22 Hz); IR (neat) 3057,2924, 2122, 1635, 1487, 1476, 1452, 1336, 1283, 123, 1212, 1190, 1090,960, 868 cm⁻¹; MS (ESI): m/z=246.07 calc. for C15H10FN[M+Na]+, found246.00.

(E)-1-(3-fluorostyryl)-2-isocyanobenzene (19)

¹H NMR (500 MHz, CDCl3) δ 7.76 (dd, J=7.8, 1.5 Hz, 1H), 7.42-7.25 (m,7H), 7.16 (d, J=16.3 Hz, 1H), 7.01-7.04 (m, 1H); ¹³C NMR (125 MHz,CDCl3) δ 167.3, 163.2 (d, J=244 Hz), 138.7 (d, J=7.3 Hz), 133.2, 131.5,130.3 (d, J=8 Hz), 129.5, 128.4, 127.3, 125.6, 125.1, 123.5, 122.8,115.4 (d, J=21 Hz), 113.5 (d, J=22 Hz); IR (neat) 3072, 3035, 2122,1608, 1581, 1482, 1448, 1286, 1238, 1183, 1158, 941, 874, 864, 834 cm⁻¹;MS (ESI): m/z=246.07 calc. for C15H10FN, found 246.00.

(E)-1-isocyano-2-(4-(trifluoromethyl)styryl)benzene (20)

¹H NMR (500 MHz, CDCl3) δ 7.75 (dd, J=7.9, 1.3 Hz, 1H), 7.70-7.61 (m,4H), 7.50 (d, J=16.3 Hz, 1H), 7.42-7.41 (m, 2H), 7.32 (t, J=6.8 Hz, 1H),7.22 (d, J=16.3 Hz, 1H); ¹³C NMR (125 MHz, CDCl3) δ 167.5, 139.8, 133.0,131.0, 130.2 (q, J=32 Hz), 129.5, 128.7, 127.4, 127.1, 125.8 (q, J=4Hz), 125.6, 125.1, 124.7, 124.1 (q, J=270 Hz); IR (neat) 2924, 2123,1614, 1483, 1321, 1190, 1155, 1104, 1065, 989, 964, 839 cm⁻¹; MS (ESI):m/z=296.07 calc. for C16H10FN[M+Na]+, found 295.84.

(E)-1-isocyano-2-(3-(trifluoromethyl)styryl)benzene (21)

¹H NMR (500 MHz, CDCl3) δ 7.82-7.77 (m, 3H), 7.61-7.45 (m, 5H), 7.36 (t,J=7.5 Hz, 1H), 7.25 (d, J=16.3 Hz, 1H); ¹³C NMR (125 MHz, CDCl3) δ167.3, 137.2, 133.1, 131.3 (q, J=31 Hz), 131.1, 129.6, 129.5, 129.3,128.6, 127.4, 125.6, 125.02, 124.99, 124.0, 124.0 (q, J=271 Hz), 123.9;IR (neat) 3049, 2118, 1489, 1341, 1324, 1287, 1224, 1163, 1194, 1114,1093, 998, 983, 962 cm⁻¹; MS (ESI): m/z=296.07 calc. forC16H10FN[M+Na]+, found 296.08.

(E)-1-isocyano-2-(2-(trifluoromethyl)styryl)benzene (22)

¹H NMR (500 MHz, CDCl3) δ 7.86 (d, J=7.8 Hz, 1H), 7.76 (d, J=7.9 Hz,1H), 7.70 (d, J=7.9 Hz, 1H), 7.61-7.54 (m, 2H), 7.45-7.38 (m, 4H), 7.33(td, J=8.0, 1.3 Hz, 1H); ¹³C NMR (125 MHz, CDCl3), δ 167.2, 135.4,133.2, 132.1, 129.6, 128.7, 128.3, 128.1, 127.9 (q, J=29 Hz), 127.5,127.3, 126.1, 126.0 (q, J=5.4 Hz), 125.9, 125.3, 124.3 (q, J=269 Hz); IR(neat) 3065, 2926, 2125, 1490, 1311, 1291, 1227, 1202, 1060, 1033, 959cm⁻¹; MS (ESI): m/z=296.07 calc. for C16H10F3N[M+Na]+, found 296.16.

(E)-1-isocyano-2-(4-methylstyryl)benzene (23)

¹H NMR (500 MHz, CDCl3) δ 7.75 (dd, J=7.6, 1.1 Hz, 1H), 7.48 (d, J=8.1Hz, 2H), 7.35-7.41 (m, 3H), 7.26 (td, J=7.4, 1.2 Hz, 1H), 7.20 (d, J=7.1Hz, 2H), 7.18 (d, J=16.0 Hz, 1H), 2.38 (s, 3H); ¹³C NMR (125 MHz, CDCl3)δ 166.8, 138.7, 133.9, 133.6, 132.6, 129.5, 129.4, 127.7, 127.3, 126.9,125.3, 124.8, 121.1, 21.3; IR (neat) 3023, 2919, 2115, 1630, 1510, 1478,1445, 1290, 1110, 959, 839 cm⁻¹; MS (ESI): m/z=242.09 calc. forC16H13N[M+Na]+, found 242.00.

(E)-1-(4-butylstyryl)-2-isocyanobenzene (24)

¹H NMR (500 MHz, CDCl3) δ 7.73 (d, J=8.0 Hz, 1H), 7.50 (d, J=7.9 Hz,2H), 7.36-7.30 (m, 3H), 7.26 (t, J=7.4 Hz, 1H), 7.22 (d, J=7.5 Hz, 2H),7.19 (d, J=15.8 Hz, 1H), 2.64 (t, J=7.8 Hz, 2H), 1.63 (m, 2H), 1.39 (m,2H), 0.95 (t, J=7.4 Hz, 3H); ¹³C NMR (125 MHz, CDCl3) δ 166.9, 143.8,134.0, 133.9, 132.7, 129.4, 128.9, 127.8, 127.3, 127.0, 125.4, 124.8,121.2, 35.5, 33.6, 22.4, 14.0; IR (neat) 2956, 2928, 2857, 2116, 1736,1632, 1608, 1480, 1449, 1265, 1241, 1018, 962, 854 cm⁻¹; MS (ESI):m/z=284.14 calc. for C19H19N[M+Na]+, found 284.16.

(E)-1-isocyano-2-(4-nitrostyryl)benzene (25)

¹H NMR (500 MHz, CDCl3) δ 8.25 (d, J=11.7 Hz, 2H), 7.76 (d, J=10.5 Hz,1H), 7.70 (d, J=11.7 Hz, 2H), 7.56 (d, J=21.8 Hz, 1H), 7.30-7.46 (m,3H), 7.24 (d, J=21.8 Hz, 1H); ¹³C NMR (125 MHz, CDCl3) δ 167.8, 147.4,142.7, 132.5, 130.2, 129.6, 129.2, 127.5 (2C), 126.6, 125.8, 124.2,123.7; IR (neat) 2923, 2842, 2121, 1632, 1510, 1372, 1340, 1299, 1269,1253, 1226, 1172, 1108, 1091, 1026, 959, 886, 870 cm⁻¹; MS (ESI):m/z=273.06 calc. for C15H10N2O2[M+Na]+, found 273.12.

1-(cyclohexylidenemethyl)-2-isocyanobenzene (26)

¹H NMR (500 MHz, CDCl3) δ 7.36-7.34 (m, 1H), 7.32 (d, J=7.5 Hz, 1H),7.26 (d, J=7.5 Hz, 1H), 7.27-7.20 (m, 1H), 2.33 (t, J=6.1 Hz, 2H), 2.25(t, J=5.6 Hz, 2H), 1.70-1.52 (m, 6H); ¹³C NMR (125 MHz, CDCl3) δ 165.2,147.2, 135.3, 130.3, 128.6, 126.7, 126.6, 125.8, 116.9, 37.3, 29.8,28.4, 27.7, 26.4; IR (neat) 2927, 2853, 2118, 1479, 1461, 1445, 1343,1038, 838 cm⁻¹; MS (ESI): m/z=220.11 calc. for C14H15N[M+Na]+, found220.08.

(E)-2-(2-isocyanostyryl)pyridine (27)

¹H NMR (500 MHz, CDCl3) δ 8.65-8.63 (m, 1H), 7.88 (d, J=16.2 Hz, 1H),7.80-7.76 (m, 1H), 7.71 (td, J=7.7, 1.8 Hz, 1H), 7.53 (dt, J=7.9, 1.1Hz, 1H), 7.45-7.38 (m, 2H), 7.35-7.28 (m, 2H), 7.21 (ddd, J=7.6, 4.8,1.1 Hz, 1H); ¹³C NMR (125 MHz, CDCl3) δ 167.6, 154.9, 149.9, 136.6,133.1, 132.4, 129.5, 128.7, 127.5, 126.3, 126.1, 125.3, 122.8, 122.0; IR(neat) 3075, 3042, 2118, 1581, 1560, 1485, 1469, 1453, 1427, 1331, 1303,1279, 1239, 1209, 1180, 1149, 1091, 1049, 992, 965 1, 897, 889, 862cm⁻¹; MS (GC-MS) m/z=207.08 calc. for C14H10N2[M+H]+, found 207.1.

(E)-2-(3-isocyanostyryl)pyridine (28)

¹H NMR (500 MHz, CDCl3) δ 8.62 (ddd, J=4.8, 1.8, 0.9 Hz, 1H), 7.69 (td,J=7.7, 1.8 Hz, 1H), 7.64-7.55 (m, 3H), 7.43-7.35 (m, 2H), 7.32-7.28 (m,1H), 7.20 (td, 1H, J=4.8, 1.2 Hz), 7.17 (d, J=16.1 Hz, 1H); ¹³C NMR (125MHz, CDCl3) δ 164.2, 154.7, 149.9, 138.4, 136.7, 130.4, 130.1, 129.8,128.0, 127.1, 125.8, 124.5, 122.7 (2C); IR (neat) 3065, 3002, 2925,2131, 1597, 1583, 1560, 1471, 1441, 1430, 1334, 1302, 1275, 1240, 1209,1146, 1094, 1083, 978, 951, 892, 863 cm⁻¹; MS (GC-MS) m/z=207.08 calc.for C14H10N2[M+H]+, found 207.1.

(E)-2-(4-isocyanostyryl)pyridine (29)

¹H NMR (500 MHz, CDCl3) δ 8.62 (ddd, J=4.8, 1.8, 0.9 Hz, 1H), 7.69 (td,J=7.7, 1.8 Hz, 1H), 7.63 (d, J=16.1 Hz, 1H), 7.60-7.57 (m, 2H),7.40-7.36 (m, 3H), 7.22-7.16 (m, 2H); ¹³C NMR (125 MHz, CDCl3) δ 164.8,154.8, 149.8, 137.9, 136.7, 130.8, 130.1, 127.8 (2C), 126.8 (2C), 122.7(2C), 122.7; IR (neat) 3056, 2922, 2851, 2130, 1583, 1562, 1505, 1468,1430, 975 cm⁻¹; MS (GC-MS) m/z=207.08 calc. for C14H10N2[M+H]+, found207.1.

(E)-3-(2-isocyanostyryl)pyridine (30)

¹H NMR (500 MHz, CDCl3) δ 8.74 (d, J=2.3, 1H), 8.55 (dd, J=4.8, 1.6 Hz,1H), 7.95-7.92 (m, 1H), 7.77-7.73 (m, 1H), 7.46 (d, 1H, J=16.3 Hz),7.44-7.38 (m, 2H), 7.36-7.29 (m, 2H), 7.18 (d, 1H, J=16.3 Hz); ¹³C NMR(125 MHz, CDCl3) δ 167.5, 149.5, 149.2, 133.0, 132.9, 132.1, 129.6,128.9, 128.7, 127.4, 125.6, 125.1, 124.4, 123.7; IR (neat) 3033, 2119,1583, 1565, 1485, 1450, 1423, 1278, 1228, 1184, 1161, 1091, 1043, 1022,962, 909 cm⁻¹; MS (GC-MS) m/z=207.08 calc. for C14H10N2[M+H]+, found207.1.

(E)-3-(3-isocyanostyryl)pyridine (31)

¹H NMR (500 MHz, CDCl3) δ 8.74 (dd, J=2.3, 0.9 Hz, 1H), 8.53 (dd, J=4.7,1.6 Hz, 1H), 7.84 (ddd, J=8.0, 2.3, 1.6 Hz, 1H), 7.56-7.52 (m, 2H),7.44-7.38 (m, 1H), 7.34-7.28 (m, 2H), 7.11 (s, 2H); ¹³C NMR (125 MHz,CDCl3) δ 164.4, 149.3, 148.7, 138.3, 132.9, 132.2, 129.9, 128.5, 127.5,127.4, 127.2, 125.7, 124.2, 123.7; IR (neat) 3027, 2124, 1598, 1581,1567, 1481, 1435, 1411, 1273, 1182, 1024, 966, 885 cm⁻¹; MS (GC-MS)m/z=207.08 calc. for C14H10N2[M+H]+, found 207.1.

(E)-3-(4-isocyanostyryl)pyridine (32)

¹H NMR (500 MHz, CDCl3) δ 8.73 (d, J=2.2 Hz, 1H), 8.53 (dd, J=4.8, 1.6Hz, 1H), 7.83 (dt, J=8.0, 2.0 Hz, 1H), 7.57-7.51 (m, 2H), 7.41-7.36 (m,2H), 7.31 (ddd, J=8.0, 4.8, 0.9 Hz, 1H), 7.11 (d, J=4.3 Hz, 2H); ¹³C NMR(125 MHz, CDCl3) δ 164.9, 149.2, 148.7, 137.9, 132.9, 132.2, 128.9,127.4 (2C), 127.3, 126.9 (2C), 123.6; IR (neat) 3028, 2920, 2850, 2127,1644, 1600, 1574, 1567, 1503, 1483, 1421, 1409, 1329, 1304, 1252, 1166,1130, 1100, 1022.75, 964, 942, 865 cm⁻¹; MS (GC-MS) m/z=207.08 calc. forC14H10N2[M+H]+, found 207.1.

(E)-4-(2-isocyanostyryl)pyridine (33)

KKB-1-19: ¹H NMR (500 MHz, Benzene-d6) δ 8.54-8.46 (m, 2H), 7.40 (d,J=16.3 Hz, 1H), 7.01 (dd, J=8.0, 1.3 Hz, 1H), 6.82-6.71 (m, 4H), 6.60(td, J=7.7, 1.4 Hz, 1H), 6.49 (d, J=16.3 Hz, 1H); ¹³C NMR (125 MHz,CDCl3) δ 167.8, 150.4 (2C), 143.6, 132.5, 130.1, 129.6, 129.2, 127.5,126.6, 125.9, 125.3, 121.1 (2C); IR (neat) 3052, 2922, 2852, 2122, 1592,1549, 1495, 1479, 1451, 1413, 1309, 1274, 1243, 1213, 1090, 991, 966,958, 880 cm⁻¹; MS (GC-MS) m/z=207.08 calc. for C14H10N2[M+H]+, found207.1.

(E)-4-(3-isocyanostyryl)pyridine (34)

¹H NMR (500 MHz, CDCl3) δ 8.64-8.58 (m, 2H), 7.58-7.52 (m, 2H), 7.42 (t,J=8.1 Hz, 1H), 7.38-7.35 (m, 2H), 7.34-7.31 (m, 1H) 7.23 (d, J=16.3 Hz,1H), 7.05 (d, J=16.3 Hz, 1H); ¹³C NMR (125 MHz, CDCl3) δ 164.7, 150.4(2C), 143.7, 137.8, 130.8, 129.9, 128.4, 127.8, 127.3, 126.2, 124.5,121.0 (2C); IR (neat) 3056, 3029, 2922, 2128, 1591, 1549, 1494, 1478,1450, 1415, 1242, 1217, 1174, 991, 971, 922, 893, 874, 859 cm⁻¹; MS(GC-MS) m/z=207.08 calc. for C14H10N2[M+H]+, found 207.1.

(E)-4-(4-isocyanostyryl)pyridine (35)

¹H NMR (500 MHz, CDCl3) δ 8.64-8.59 (m, 2H), 7.60-7.53 (m, 2H),7.42-7.35 (m, 4H), 7.26 (d, 1H, J=16.3 Hz), 7.04 (d, J=16.3 Hz, 1H); ¹³CNMR (125 MHz, CDCl3) δ 165.3, 150.4 (2C), 143.8, 137.3, 131.1, 128.4,127.8 (2C), 126.9 (2C), 126.2, 121.0 (2C); IR (neat) 3031, 2923, 2118,1582, 1559, 1504, 1486, 1469, 1453, 1427, 1331, 1302, 1279, 1239, 1209,1180, 1148, 1091, 992, 962, 942, 898, 858 cm⁻¹; MS (GC-MS) m/z=207.08calc. for C14H10N2[M+H]+, found 207.1.

(E)-1-isocyano-3-styrylbenzene (36)

¹H NMR (500 MHz, CDCl3) δ 7.54-7.50 (m, 4H), 7.41-7.35 (m, 3H),7.34-7.28 (m, 1H), 7.26-7.23 (m, 1H), 7.14 (d, J=16.3 Hz, 1H), 7.04 (d,J=16.3 Hz, 1H); ¹³C NMR (125 MHz, CDCl3) δ 164.1, 139.0, 136.4, 131.1,129.7, 128.8 (2C), 128.4, 127.4, 127.1, 126.8 (2C), 126.4, 125.1, 124.0;IR (neat) 3024, 2922, 2126, 1597, 1578, 1496, 1485, 1472, 1449, 1267,1226, 1168, 1145, 1081, 1074, 965, 911, 884 cm⁻¹; MS (GC-MS) m/z=206.09calc. for C15H11N[M+H]+, found 206.1.

(E)-1-isocyano-4-styrylbenzene (37)

¹H NMR (500 MHz, CDCl3) δ 7.54-7.49 (m, 4H), 7.41-7.35 (m, 4H),7.33-7.28 (m, 1H), 7.16 (d, J=16.3 Hz, 1H), 7.08 (d, J=16.3 Hz, 1H); ¹³CNMR (125 MHz, CDCl3) δ 164.5, 138.6, 136.5, 131.1 (2C), 128.8 (2C),128.4 (2C), 127.2 (2C), 126.8 (3C), 125.2; IR (neat) 3023, 2920, 2850,2123, 1633, 1576, 1503, 1448, 1417, 1335, 1305, 1220, 1198, 1160, 1107,1073, 967, 950, 918, 866 cm⁻¹; MS (GC-MS) m/z=206.09 calc. forC15H11N[M+H]+, found 206.1.

2-isocyano-5-methyl-4′-(trifluoromethyl)-1,1′-biphenyl (50)

Prepared according to a procedure reported by Studer (Zhang, B. et al.,Angew. Chem., Int. Ed. 2013, 52, 10792) from 2-bromo-4-methylanaline and4-(trifluoromethyl)phenylboronic acid. ¹H NMR (500 MHz, CDCl3) δ 7.77(d, J=8.1 Hz, 2H), 7.66 (d, J=8.1 Hz, 2H), 7.44 (d, J=8.6 Hz, 1H),7.26-7.27 (m, 2H), 2.47 (s, 3H); 13C NMR (125 MHz, CDCl3) 166.8, 140.9,140.4, 137.3, 131.1, 130.4 (q, J=32 Hz), 129.8, 129.6, 127.9, 125.7,124.2 (q, J=270 Hz), 122.3, 21.3; IR (cm-1): 2922, 2125, 1620, 1571,1493, 1396, 1328, 1198, 1179, 1155, 1110, 965, 952, 897, 880, 845; MS(ESI): m/z=262.09 calc. for C15H10F3N[M+H]+, found 262.2.

1-isocyano-2-phenethylbenzene (53)

¹H NMR (500 MHz, CDCl3) δ 7.37 (dd, J=7.8, 1.4 Hz, 1H), 7.33-7.28 (m,3H), 7.27-7.19 (m, 5H), 3.07 (dd, J=8.9, 7.6 Hz, 2H), 2.95 (dd, J=8.9,5.6 Hz, 2H); ¹³C NMR (125 MHz, CDCl3) δ 166.1, 140.8, 138.2, 130.0,129.4, 128.6 (2C), 128.5 (2C), 127.1, 126.9, 126.2, 126.1, 36.0, 34.7;IR (neat) 3063, 3028, 2926, 2855, 2119, 1603, 1496, 1487, 1453 cm⁻¹; MS(GC-MS) m/z=208.11 calc. for C15H13N[M+H]+, found 208.1.

Compounds 46, 49 and 51 were prepared according to the literatureprocedure (Zhang, B. et al., Angew. Chem., Int. Ed. 2013, 52, 10792) andtheir spectral data match with the reported ones. Compounds 5, 6, 11(Zhang, B. & Studer, A., Org. Lett. 2014, 16, 1216), 42 (Tanaka, R. etal., J. Am. Chem. Soc. 2008, 130, 2904), and 43 (Hahn, B. T. et al.,Angew. Chem., Int. Ed. 2010, 49, 1143) were prepared according to therepresentative procedure aforementioned and their spectral data matchwith the reported ones.

Biological Materials and Evaluation Methods:

Bacterial Strains and Reagents.

Clinical isolates of MRSA, vancomycin-intermediate S. aureus (VISA), andvancomycin-resistant S. aureus (VRSA) were obtained through the Networkof Antimicrobial Resistance in Staphylococcus aureus (NARSA) program(FIG. 6). Vancomycin hydrochloride (Gold Biotechnology, St. Louis, Mo.,USA) and linezolid (Chem-Impex International, Inc., Wood Dale, Ill.,USA) powders were purchased commercially and dissolved in DMSO toprepare a stock 10 mM solution.

Assessment of Antimicrobial Activity of the Isonitrile Compounds AgainstMultidrug-Resistant S. aureus Strains.

The minimum inhibitory concentration (MIC) of each compound andlinezolid was determined against eight different strains of MRSA, VISA,and VRSA using a modified version of the broth microdilution method,outlined by the CLSI. Institute CaLS, Methods for Dilution AntimicrobialSusceptibility Tests for Bacteria That Grow Aerobically—Seventh Edition:Approved Standard M7-A7. 7 ed. Wayne, Pa. 2011. The same analysis wasperformed with vancomycin against the VISA and VRSA strains tested. Abacterial suspension (˜1 Å˜105 CFU/mL) was prepared in Tryptic soy broth(TSB) and then transferred to a microtiter plate. Each agent tested wasadded (in triplicate) to wells in the first row of the plate and thenserially diluted downward. Plates were incubated at 37° C. for 18-20 hbefore the MIC was determined as the lowest concentration of each testagent where bacterial growth was not visible.

Toxicity Analysis of Selected Isonitrile Compounds Tested AgainstMammalian Cells.

Selected isonitrile compounds were assayed at concentrations of 16 μM,32 μM, 64 μM, and 128 μM against a murine macrophage (J774) cell line toassess if the compounds exhibited toxicity to mammalian cells in vitro.Cells were cultured in Dulbeco's modified Eagle's medium (Sigma-Aldrich,St. Louis, Mo., USA) with 10% fetal bovine serum (USA Scientific, Inc.)at 37° C. with 5% CO2. Controls received DMSO alone at a concentrationequal to that in drug-treated cell samples. The cells were incubatedwith each compound (in triplicate) in a 96-well tissue-culture plate at37° C. and 5% CO2 for 2 h prior to addition of the assay reagent MTS3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)(Promega, Madison, Wis., USA). Absorbance readings (at OD490) were takenusing a kinetic microplate reader (Molecular Devices, Sunnyvale, Calif.,USA). The quantity of viable cells after treatment with each compoundwas expressed as a percentage of the viability of DMSO-treated controlcells (average of triplicate wells±standard deviation). Statisticalanalysis was performed (comparing cells treated with compound versuscells treated with DMSO) using the paired t-test (P<0.05) utilizingMicrosoft EXCEL software.

Caco-2 Permeability Analysis of Compound 13.

The ability of compound 13, ranitidine (low permeability control),warfarin (high permeability control), and talinolol (P-glycoproteinefflux substrate), to effectively permeate across a biological membranewas assessed using a Caco-2 cell monolayer, as described elsewhere.Mohammad, H. et al., J. Antibiot. 2014, doi:10.1038/ja.2014, 142. Theamount of permeation was determined both from the apical (A) tobasolateral (B) direction and the basolateral (B) to apical (A)direction. Data for apparent permeability (Papp) and the efflux ratio(RE) were determined as explained elsewhere. Mohammad, H. et al., ibid.An RE>2 indicates the test agent may be a potential substrate forP-glycoprotein or other active efflux transporters.

Kinetic Solubility Screen.

A kinetic solubility analysis of compound 13, reserpine, tamoxifen, andverapamil was performed as has been described elsewhere. Mohammad, H. etal., ibid. The solubility limit (in μM) reported is the maximumconcentration of each test agent where turbidity was not observed.Values below 1 μM indicate compound is insoluble, values between 1 to100 μM indicate partial aqueous solubility, and values above 100 μMindicate test agent is fully soluble.

Metabolic Stability Analysis Using Pooled Human Liver Microsomes.

To analyze the stability of compound 13 to metabolic processes in theliver, this compound was incubated in duplicate with pooled human livermicrosomes at 37° C. (for 60 min), using a similar protocol describedelsewhere, with two modifications. Zhang, W. et al., Bioorgan. Med.Chem. 2012, 20, 1029; Papadopoulou, M. V. et al., Future Med. Chem.2013, 5, 1763. First, the reaction mixture utilized 0.3 mg/mL microsomalprotein. Additionally, samples were collected after 0 and 60 min andanalyzed accordingly. Data are reported as % remaining by dividing bythe time zero concentration value.

It should be understood, of course, that the foregoing relates toexemplary embodiments of the invention and that modifications may bemade without departing from the spirit and scope of the invention as setforth in the following claims.

We claim:
 1. An isonitrile compound having the structure:

wherein R is a hydrogen, an alkyl, a cycloalkyl, a heteroalkyl, or anaryl group; n is an integer from 0 to 4; X is hydrogen, a C1 to C3 alkylgroup, a halogen, alkyloxy, trihalomethyl, or a nitro group; W, Y and Zare each independently —CH—, or —N—; and pharmaceutically acceptablesalts thereof.
 2. The compound of claim 1, wherein W, Y and Z are —CH—.3. The compound of claim 1, wherein R is methyl, ethyl, n-propyl,n-butyl or phenyl.
 4. The compound of claim 1, wherein n is
 0. 5. Thecompound of claim 1 wherein X is —F, —CF₃, —OMe, n-butyl, or —NO₂.
 6. Amethod for inhibiting MRSA comprising contacting MRSA with theisonitrile compound of claim
 1. 7. The method of claim 6, wherein thestrain of MRSA is NRS1, NRS72, NRS119, NRS382, NRS383, NRS384, NRS385,NRS386, VRS2, or any combination thereof.
 8. A method for treating apatient having a MRSA infection comprising administering to the patienta therapeutically effective amount of the isonitrile compound ofclaim
 1. 9. The method of claim 8, wherein the isonitrile compound isadministered with a second antibacterial compound.